In the last 50 years, and especially in the last decade, there has been a remarkable trend towards both the automation and the miniaturization of chemical analysis and electromechanical systems. The limits of detection of primary analytical methods have improved by many quantum leaps. Mass spectrometry can detect attomoles of sample using nanospray methods. Nuclear Magnetic Resonance (NMR) can now detect pmoles of analyte using 5 nL microcoil probes (Olson, 1995), a 500-fold improvement over 1980's technology. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can detect zeptomoles of analyte in a volume of picoliters. The tremendous sensitivity of these microscale analytical technologies, however, is useless without the ability to efficiently load and deliver the appropriate microscale samples. For example, capillary electrophoresis analysis generally requires providing several microliters of sample, from which a few nanoliters is drawn. Loading a 1 μL microcoil NMR probe requires filling a 10 μL dead volume. On microfluidic chips, samples typically are introduced to fill entire channels, of which only a small segment may occupy a region of detection or be injected into a separation channel.
An obvious alternative would be to supply small samples and drive them through the conduit either with air or with clean solvent. However, in pressure-driven liquid flow, a sample originating as a short volume segment of a conduit will disperse into a larger volume, with concomitant dilution, proportionally to the volume through which it is moved: the boundary layer at the conduit wall is immobile; however, flow at the center of the conduit is rapid. Although dispersion of small concentrated samples can be significant even within the few-cm distances of a microfluidic chip, the problem is most vividly defined and discussed in the example of flow-NMR, where samples must be transported over distances of several meters.
NMR is a very information-rich spectroscopy, well-established for confirming the structure and purity of newly synthesized compounds or isolated natural products. It has also proven valuable in metabonomics, using pattern recognition software to analyze large numbers of complex spectra. However, the low sensitivity of NMR (1000-fold less than mass spectrometry) is problematic, particularly in LC-NMR where acquisition time is limited and compounds of interest may be a small fraction of the permissible column load. NMR sensitivity has been improved modestly (2-4-fold) using higher field magnets and cryogenically-cooled electronics (cryoprobes). For mass-limited samples, microcoil NMR probes offer up to a 500-fold sensitivity increase (Olson, 1995), however, efficiently loading microcoil probes is a challenge (Kautz, 2001). The detection cell has a volume of 30 nL to 1 μL is recessed 50 cm or more up the narrow bore of the NMR magnet. In contrast to conventional NMR probes, the microcoil probe's axis is oriented transverse to the magnet bore so sample tubes cannot be inserted without removing the probe, and consequently microcoil probes are generally implemented as a flow cell. An additional complication is that any motorized equipment must be located outside the magnet's fringe field, necessitating an additional 1-10 meters of capillary tubing, depending on the magnet's fringe field. The current commercial offering is a compromise with these limitations, using the smallest feasible transfer capillaries to fill a relatively large flowcell. But the challenge of filling a 1 μL observed volume in a 5 μL flow cell through several meters of 50 micron capillary tubing (2 μL/meter) has severely limited microcoil NMR's sensitivity in practice.
The two traditional approaches to flow-NMR (Keifer, 2003a) are direct-injection NMR (Keifer, 2000) and flow-injection analysis-NMR (Keifer, 2003b). The methods differ in how they optimize the necessary steps of clearing, washing and reloading the NMR probe flow cell through the 2-5 meter transfer line while avoiding sample dilution in the dead volumes of the transfer line and NMR probe flow cell. In direct injection NMR, samples are injected into an empty (air-filled) flow cell through a 100 μm i.d. or larger transfer line. Samples can be injected relatively quickly without dilution; however, the percentage of the injected sample that ultimately resides within the NMR coil observed volume during spectral analysis, is low. The need for a wash cycle to reduce sample-to-sample carryover to <1% increases the sample change time. Because it is not feasible to flush 50 micron capillaries longer than 1 meter with air, and larger capillaries have a prohibitively large volume, direct injection methods have only been implemented on microcoil probes manually. Working at the closest approach to the magnet bore, it is possible to fill the flow cell using 8 μL samples.
In flow injection NMR, the flow lines are maintained filled with solvent. Samples are introduced by means of a sample loop valve and delivered to the probe by a liquid chromatographic pump. Because the sample disperses into the carrier solvent during transfer, the final analyte concentration in the NMR coil depends on the sample volume, flow rate, and system dead volume. Sharp gradients of analyte concentration near the NMR coil immediately after injection can cause poor line shape, and an equilibration time of 1-2 min may be required for line shape to sharpen as the analyte diffuses throughout the flow cell. Because the effects of these gradients are more pronounced for dissimilar solvents, the same solvent must be used for both the carrier and sample preparation.
FIA-NMR methods are applicable to microcoil probes, and a high-throughput FIA-NMR method using a commercial microcoil probe with a microfluidic sample loader (Olson, 2004) has been introduced. This method requires 10 μL of sample to deliver at full concentration, or dilutes smaller samples to a dead volume of 10 μL in the course of loading. 50 μL of deuterated solvent was also required per sample to reduce carryover below 1%.
Another approach is segmented flow, in which an immiscible fluid is used to push a small sample as a bolus or “plug” through the fluidic conduit. This approach appears to offer several advantages. Smaller samples could be used, so sample consumption would be lower. Samples would not be diluted, so NMR acquisition time would be faster. Samples could be more accurately positioned in the detection cell, so setup would be straightforward, faster and provide better sensitivity. There would be no “equilibration time” required for lineshape to improve after injection. And for high-throughput operation, a queue of samples could be quickly advanced a short distance, rather than having each new sample delivered the entire conduit distance. While the stability of segmented plugs in the 3-mm vertical flow cells of conventional saddle coil LC/NMR probes is problematic, several preliminary findings with segmented plugs in microcoil NMR probes appear promising. Segmented flow has historically been implemented in clinical analyses and has recently been demonstrated in a microfluidic chip (Ramsey, 2003).
In work on the optimal sizes of microcoil probes, it was shown that samples sandwiched on both sides by the immiscible fluorocarbon fluid FC 43 could be much smaller than samples sandwiched between air bubbles without degradation of the NMR line shape: only twice the coil size instead of 7 times (Behnia, 1998). The utility of this fluorocarbon bracketing was demonstrated in obtaining spectra from the 500-ng eluate of a single solid-phase synthesis bead (Lacey, 2001).
However, substantial challenges remain in putting this approach into practice (Macnaughtan, 2003). Sample plugs are frequently lost or degraded in a variety of ways. Principally, moving sample plugs leave a film of solvent on the wall of the conduit, e.g., capillary, and this film can consume about 2 μL of sample per meter of movement, which is completely prohibitive. All of this lost material can mix with subsequent plugs, resulting in high carryover (Patton et al., 1997). Plugs also have tended either to acquire large discrete breaks in the middle (“fragmentation”) or to form many small breaks (“frothing”) at their ends. Both of these effects increased with increasing capillary size, where the outward pressure of the curved surface of the plug was insufficient to hold the plug against the conduit wall in capillaries over 300 μm diameter. Improvements in these techniques would be greatly appreciated.